The present invention relates to methods that will allow the measurement of total circulating red cell volume, total blood volume and red cell survival, and, in particular, to making such measurements without exposing the subject to radiation. This invention allows multiple measurements in the same individual over clinically meaningful periods of time (e.g., 72 hours). The process requires only readily available equipment, such as a gamma counter. The process is sensitive enough for potential application even to a low birth weight infant or fetus.
All methods for measuring red cell volume in common use depend upon the measurement of the initial dilution of erythrocytes, or red blood cells, labeled with one of a variety of tracers, such as radionuclides. Typically, a volume of blood is drawn from the subject, the red blood cells are labeled with a tracer, and reinjected into the subject where they are diluted in the entire volume of the subject's blood. At a later time, one or more samples of blood are withdrawn and the labeled blood cells are quantified. A straightforward calculation then yields the total volume of red blood cells. Likewise, total blood volume may be calculated from the red cell volume and hematocrit.
Commonly the tracer used to label the red blood cells is a radionuclide. The standard radionuclide used is .sup.51 Cr because the red cell binding properties of chromium are excellent, though not perfect. Other radionuclides have also been investigated as red cell tracers; these nuclides include .sup.99M Tc and .sup.111 In. These two radionuclides have the desirable characteristic of short half-lives leading to reduced radiation exposure; however, these same short half-lives mandate a readily available supply and predictable timing for the use of the radionuclide. Each of these elements has its own special problems in conversion to chemical forms that will bind firmly to red cells. Short half lives also complicate and limit the measurement of red cell survival.
The special binding properties of chromium have been explored in an attempt to develop a practical, nonradioactive method for determination of red cell volume. One method uses .sup.50 Cr, a stable isotope of chromium, with subsequent neutron activation. Another uses .sup.52 Cr, the abundant stable chromium isotope, with atomic absorption analysis. A third uses cesium with subsequent analysis of x-ray stimulated fluorescence. Each of these methods requires highly specialized equipment: either 1) a neutron source, 2) a Zeeman electrothermal atomic absorption spectrometer, or 3) an .sup.247 Americium source and highly sensitive 1024-channel silicon detector.
Plasma volume can be measured based on the dilution of labeled albumin or dyes that bind to albumin such as Evan's blue dye; blood volume and red cell volume can then be calculated from the hematocrit; i.e., the volume after centrifugation of the cellular elements of blood in relation to the total volume. The dye methods and albumin methods both measure the albumin space and are limited by the same problems with capillary permeability. Dye dilution methods do not yield reproducible results due to variability between individuals in both mixing time and loss from circulation. These estimates are further confounded in situations of increased capillary permeability such as thermal burn, sepsis, and prematurity; in such situations, the albumin distribution volume can substantially exceed the true plasma volume.
Red cell volume can be measured once using .sup.51 Cr labeling of autologous red cells, and total blood volume can be calculated from the hematocrit. For serial measurements, which are necessary, for example, to calculate red cell survival, one must roughly double the dose of .sup.51 Cr with each successive measurement in order to produce a meaningful increment over the residual .sup.51 Cr left in the blood from the previous measurements. Thus, serial measurements result in increasing exposure to radioactivity. This problem appears to render multiple blood volume assessments by the .sup.5 Cr method impractical; indeed, a clinical application of the type proposed here has not been published despite the fact that .sup.5 Cr has been an established red cell label for more than 20 years.
Biotin is a water-soluble vitamin generally classified in the B group. A number of biotin-binding proteins are known, but avidin and streptavidin are two proteins with very high binding affinities for biotin. One is found in egg white, and the other in the secretions of the mold Streptomyces avidini. In the natural setting, both probably act as antimicrobials by preventing microbes from obtaining biotin. The equilibrium binding constant of biotin for avidin (and of biotin for streptavidin) is 10.sup.15 M.sup.-1. This extraordinarily large binding constant, as well as the large association rate constant and small dissociation rate constant, dictate that the binding is very rapid and essentially irreversible. Binding is highly specific with respect to the structure of the biotin bicyclic ring and is promoted by the hydrophobic binding pocket of avidin. This binding specificity has rendered the biotin:avidin interaction highly resistant to interference by substances such as antibiotics and chemotherapeutic agents that are often present in the plasma of individuals in clinical situations. This unusually strong and specific binding has been used for a variety of applications, including ones that use plasma from patients in many clinical settings.
The biotin:avidin interaction has already been used to measure red cell volume in normal adults, I. Cavill, et al., "The Measurement of the Total Volume of Red Cells in Man: A Nonradioactive Approach using Biotin," British Journal of Haematology, 1988, and in neonates, I. R. B. Hudson, et al., "Biotin Labeling of Red Cells in the Measurement of Red Cell Volume in Preterm Infants," Pediatric Research, 1990 Those studies found a close agreement for the red cell volume by the nonradioactive method with values from the standard .sup.51 Cr method. However, the label disappeared within seven days, making measurements of red cell survival impossible and potentially making repetitive measurements of red cell volume very complicated. In addition, the method depended upon the use of a fluorescence activated cell sorter (FACS). These machines are increasingly available in tertiary medical centers and should not be considered highly specialized equipment; however, the method of the present invention uses either a simplified system for detecting the nonradioactive label that requires only a gamma counter and a centrifuge or a FACS technique that yields accuracy not applied previously to this problem. This technique for labeling and FACS detection is novel in the accuracy attained and allows sequential RCV measurements and red cell survival measurements that have not been reported before.
An additional related clinical application has similar potential for broad application--the determination of blood volume in trauma patients arriving in the emergency room. Information about extent of bleeding prior to arriving in the emergency room could potentially be available within one hour using the FACS technology of the present invention and might be life saving. Any delay needed for washing and labeling the cells could be avoided by preparing and storing "universal donor" red cells ahead of time. Prior preparation is feasible because the labeling technique described herein produces a stable label when stored in the modern red cell storage media at 4.degree. C. These universal donor labeled red cells could be stored up to the FDA mandated limit (e.g., 35 days or 42 days).
Circulating red cell volume, or simply, red cell volume (RCV) is used herein to designate the total volume of circulating red cells. Oxygen carrying capacity is directly proportional to total circulating hemoglobin; total circulating hemoglobin can be calculated from the product of the circulating red cell volume and the mean corpuscular hemoglobin concentration. Total blood volume can be calculated from the red cell volume and the hematocrit (with corrections for trapped plasma and for the difference between peripheral and central hematocrit).
Red cell survival (RCS) is defined as the percentage of transfused red cells that remain in circulation at a given point in time. To a first approximation, red cells are removed in two phases; the early "rapid removal" phase and the remaining "slow removal" phase. By implication, there must be at least two populations of cells. The first consists of cells that are damaged and are removed from circulation rapidly after transfusion. The percent survival at 24 hours after transfusion ("post-transfusion recovery") reflects this population. The second consists of cells that are damaged minimally, if at all, and are slowly removed from circulation. The time loss of 50% of the label (T.sub.50) is a commonly used parameter and reflects primarily this population; the T.sub.50 can be calculated from the first order decay constant assuming mono-exponential disappearance. The average potential life span can be determined directly or as the time to greater than 95% disappearance of the label. These parameters are measures of the slow removal phase.
In critically ill patients care, invasive monitoring and sophisticated electronic technology have enabled the clinician to determine the cardiac output, filling pressures of the right and left side of the heart, mean pulmonary and arterial pressures, and pulmonary and peripheral vascular resistance. These advances have led to a great increase in our ability to manipulate central hemodynamics, modify cardiac activity, minimize cardiac work, and maximize cardiac output. These measurements have also given us insight into and understanding of the hemodynamic changes associated with endotoxemia and shock. A critical capability that remains unavailable is accurate serial assessment of red cell volume and total blood volume. During management of major trauma, total blood volume may vary widely among individuals and in a single individual. These changes in blood volume can have major effects on central hemodynamics and, in turn, on oxygen delivery, extraction, and consumption.
If one were able to accurately and repetitively measure red cell volume and total blood volume, then resuscitation and pharmacological management of complex patients suffering from severe sepsis or hypovolemic shock of any etiology would be enhanced because accurate values for true blood loss and true blood volume would be available serially in each patient, moreover, this information would be available hours before the body's homeostatic mechanisms compensate for the blood loss by expanding the plasma volume and producing the attendant decrease in hematocrit and hemoglobin concentration. For example, adults who are burn treatment inpatients undergo clinically indicated procedures for closure of their burn wounds. Burn wound closure procedures can result in large volume blood losses. Typical blood loss estimates are between 50 cc to 150 cc of blood loss for each 1% of the patient's body surface are that must have the burn wound excised and skin grafted. Therefore, excision and grating of a 20% body surface area burn in a 70 kg adult male can result in a blood loss of 1,000-3,000 cc. This loss occurs over about one hour. Assuming a blood volume of 5,000 cc, this would represent an acute loss of 20%-60% of total blood volume. Currently intra- and peri-operative blood loss is estimated by a gross subjective examination of blood on discarded sponges and drapes. Current clinical estimates of circulating blood volume in the perioperative and postoperative patient include indirect findings such as blood pressure, pulse, urine output, the acid-base balance, extremity temperature, and skin color. Serial measurements of hemoglobin and hematocrit can also be used to estimate blood volume but actually reflect the red blood cell concentration rather than red blood cell volume. The ability to accurately determine circulating red cell volume and blood volume several times during critical periods would greatly aid in the care of the unstable and hypovolemic patient by accurately dictating red blood cell and crystalloid transfusion requirements for replacement of losses. Unfortunately, repetitive measurements are not currently feasible using available methods for the technical reasons discussed above.
Premature infants are among the most heavily transfused of all patient groups. Of the 38,000 premature neonates with birth weight greater than 1500 g who are born each year in the United States, about 80% require multiple red cell transfusions; many of these infants receive cumulative transfusion volumes in excess of their total blood volume. Multiply-transfused infants may be exposed to two to eighteen different donors. Concerns about transfusion-related disease have not caused a striking reduction in blood utilization by these infants because the transfusions are perceived to be necessary.
Despite the fact that neonatal transfusions consume important amounts of health care resources and are associated with increased risk of morbidity and mortality, the scientific basis for most neonatal transfusion practices is considerably weaker than the basis for transfusion practices in adults. An important limitation in knowledge about neonatal anemia is the inability to measure red cell survival and blood volume because current methods expose the infant to unacceptable doses of radiation.
The idea that the portion of the infant's blood retained in the placenta (so called "cord blood") could be harvested and provided to the infant at a later time has attracted attention recently. In theory, use of these autologous red cells would minimize transfusion transmitted disease. However, knowledge of the storage characteristics of this fetal blood is quite limited. In order to assess the efficacy of storage, measurement of the survival of the transfused red cells is required. However, current practical methods are precluded by the required radiation exposure.
There is also a need for a nonradioactive method for red cell volume of the pregnant woman and the fetus. Changes in red cell volume and blood volume have been hypothesized to have pathogenic roles and prognostic value in toxemia of pregnancy, gestational diabetes, and gestational exacerbation of connective tissue disease for the mother, and in intrauterine growth retardation and hydrops secondary to immune, infectious, and cardiac causes for the fetus. .sup.51 Cr measurements are precluded and, as discussed below, measurements of red cell and vascular volume by alternative methods are impractical or unsatisfactory.
The problems and disadvantages of the prior art methods and the significant advantages of a non-radioactive method of determining red cell and blood volume and red cell survival as described above are addressed by the method of the present invention. This method builds on the method originally reported by Cavil, et al. that suggested biotinylation of red blood cells and subsequent incubation in florescinated streptavidin would allow quantitation of the red blood cell's dilution in vivo using florescence activated cell sorting (FACS). The present invention also utilizes biotinylated red blood cells but with significant enhancements. The biotin label is firmly attached allowing more accurate determination of the red cell volume and blood volume. One detection system of the present invention uses .sup.25 I-Streptavidin and gamma counting as a detection system. Rather than being bound to the red blood cells injected into the patient, the radionuclide (complexed with streptavidin or other biotin-binding protein) in the present process is bound to the biotinylated red blood cells after the second volume of blood containing the diluted biotinylated cells is withdrawn from the patient. The method of the present invention is also enhanced by separation of red blood cells from other types of blood cells and plasma components using a density gradient. Thus the technology is more practical. The accuracy of the method has been demonstrated in vivo and has the capability of measuring survival red blood cells after transfusion as well--a capability not available using the method of Cavil, et al. As disclosed herein, we have made two important breakthroughs in the use of FACS technology in this application. These breakthroughs have enabled highly accurate measurements of red cell volume and red cell survival that permit completely new applications such as sequential measurement of red cell volume. A concept of using this method for multiple measurements of blood cell volume on clinically meaningful time intervals for use in measuring trauma blood loss in trauma patients is also disclosed.